DESCRIPTION: (taken from the application). The proposed research has the long-range goal of providing an understanding of how the inhaled general anesthetics interact with proteins, from structural and dynamic points of view, at the molecular level. This will be achieved using fresh approaches developed in this laboratory to detect anesthetic binding to protein targets. The primary experimental focus of the research will be synthetic four-alpha helix bundles that can be structurally modified by established protein engineering techniques. In addition, the ability of volatile general anesthetics to interact with a membrane-soluble three-a-helix bundle will be examined. These synthetic proteins will serve as simplified models for the bundles of transmembrane a-helices that are ubiquitous structural components of ion channels and neurotransmitter receptors in the central nervous system, and are the favored targets for general anesthetics at present. Our current structural understanding of membrane proteins precludes their use to precisely examine anesthetic-protein complexation. However, the proposed use of simplified, well-defined, models of the transmembrane domains of native proteins lend themselves to the direct determination of the structural features of anesthetic binding sites using various spectroscopic approaches, molecular dynamics simulations, X-ray crystallography, and. neutron diffraction. This will provide a detailed frame to evaluate hydrophobic, electrostatic and protein cavity contributions to anesthetic binding, providing insight into the relative importance of specific molecular interactions for anesthetic complexation. The consequences of anesthetic binding to protein targets will be determined using measures of protein dynamics such as fluorescence anisotropy and hydrogen exchange, with the goal of furthering our understanding of how a bound anesthetic might alter protein function. In addition, chemical denaturation circular dichroism spectroscopy will be used to determine the effect of anesthetics on global protein stability. The proposed studies build on the reported findings on halothane binding to four-alpha-helix bundles to (1) explore binding of other classes of general anesthetics (ethers and alcohols), (2) optimize the structure of the anesthetic binding site, and (2i) examine binding of anesthetics to a membrane-soluble a-helical bundle. Ultimately, the use of such model systems will provide fundamental information concerning how these important clinical compounds interact with potential target sites in the central nervous system at the molecular level, and will establish a framework for testing such associations, with natural membrane proteins.